We report a series of copper(II) artificial metallo‐nucleases (AMNs) and demonstrate their DNA damaging properties and in‐vitro cytotoxicity against human‐derived pancreatic cancer cells. The compounds combine a tris‐chelating polypyridyl ligand, di‐(2‐pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu‐DPA‐N,N' (where N,N'=1,10‐phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X‐ray crystallography and continuous‐wave EPR (cw‐EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron‐nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp) rising from 105 to 107 m−1 with increasing extent of planar phenanthrene. Cu‐DPA‐DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine–cytosine (G‐C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu‐DPA‐DPPZ, display promising anticancer activity against human pancreatic tumour cells with in‐vitro results surpassing the clinical platinum(II) drug oxaliplatin.

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Tackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenanthroline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular docking revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (− 7.9 kcal/mol) or minor (− 7.2 kcal/mol) DNA grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive ethidium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp = 2.55 × 106 M−1) than Ag-phendione (Kapp = 2.79 × 105 M−1) and phendione (Kapp = 1.33 × 105 M−1). Cu-phendione induced topoisomerase I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA injuries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by flow cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 µM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa. Graphical abstract

Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators—DPQ and DPPZ—and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.

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